Proteins related to schizophrenia and uses thereof

ABSTRACT

Presenilin Associated Membrane Protein (PAMP), and nucleic acids encoding this protein, are provided. PAMP and PAMP nucleic acids provide diagnostic and therapeutic tools for evaluating and treating or preventing neurodevelopmental and neuropsychiatric disorders. In a specific embodiment, mutations in PAMP are diagnostic for schizophrenia. The invention further relates to screening, particularly using high-throughput screens and transgenic animal models, for compounds that modulate the activity of PAMP and presenilins. Such compounds, or gene therapy with PAMP, can be used in treating neurodevelopmental and neuropsychiatric disorders, particularly schizophrenia. In addition, the invention provides PAMP mutants, nucleic acids encoding for PAMP mutants, and transgenic animals expressing PAMP mutants, which in a preferred aspect result in biochemical, morphological, or neuropsychological changes similar to those associated with schizophrenia.

This patent application claims the priority of U.S. provisional patentapplication No. 60/229,889, filed Sep. 1, 2000, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of neurological andphysiological dysfunctions associated with neuropsychiatric andneurodevelopmental diseases, especially schizophrenia. Moreparticularly, the invention is concerned with the identification ofproteins associated with neuropsychiatric and neurodevelopmentaldiseases, especially schizophrenia, and relates to methods of diagnosingthese diseases, and to methods of screening for candidate compoundswhich modulate the interaction of a certain protein, specificallyPresenilin Associated Membrane Protein (“PAMP”), with presenilinproteins.

BACKGROUND OF THE INVENTION

The origin of and causes for schizophrenia, one of the most seriousneuropsychiatric disorders, have long been sought after. A number ofstudies have suggested that schizophrenia is predominantly genetic, butit has proven difficult to show a significant genetic linkage. However,recently, a novel locus associated with inherited susceptibility toschizophrenia has been mapped to chromosome 1 q21-q22, near theanonymous DNA markers D1S1653, D1S1679, and D1S1677 (Brzustowicz et al.,2000). Furthermore, several lines of evidence, both from morphologicaland neuropsychological findings, now indicate that schizophrenia may bea disease of central nervous system development (reviewed in Stefan etal, 1997). For example, Falkai et al., 2000, provided quantitative datashowing that the positioning of neuron (pre-alpha cell) clusters wasabnormal in schizophrenia patients, supporting the theory thatschizophrenia derives from impaired brain development. Such abnormalneuron positions could, e.g., arise from failures of neuronal migrationduring fetal development.

An important pathway implicated in the development of the nervoussystem, as well as in schizophrenia, is the Notch signaling pathway.Notch is a protein receptor for inhibitory signals that shape thepattern of the nervous system, and the localization of Notch signalingis crucial for determining where neural precursor cells arise (Baker,2000). In a series of 80 British parent-offspring trios, the NOTCH4locus was highly associated with schizophrenia (Wei and Hemmings, 2000).Possible candidate sites conferring susceptibility to schizophreniaincluded an A-to-G substitution in the promoter region, and the(CTG)_(n) repeat in exon 1, of NOTCH4.

The presenilin proteins, i.e., presenilin 1 (PS1, encoded by the PS1gene) and 2 (PS2, encoded by the PS2 gene), are involved in the Notchpathway, and form a close functional relationship with Notch during cellfate determination in a variety of species (Selkoe, 2000). Several linesof evidence have suggested roles for PS1 and PS2 genes in developmental,apoptotic signaling and in the regulation of proteolytic cleavage of theβ-amyloid precursor protein (βAPP) (Levitan et al., 1995; Wong et al.,1997; Shen et al., 1997; Wolozin et al., 1996; De Strooper et al.,1998). For example, the PS1 gene is associated with migration defects inthe central nervous system of PS1−/− mice (Hartmann et al., 1999;Handler et al., 2000). In addition, a mutation in βAPP(βAPP_(Ala713Val)) has been described in one family with aschizophrenia-like illness (Jones et al., 1992), further implicating thePS1/βAPP/Notch pathways in schizophrenia and related disorders. However,just how these putative functions are mediated, and how they relate tothe abnormal metabolism of the βAPP associated with PS1 and PS2mutations remains to be elucidated (Martin et al., 1995; Scheuner etal., 1996; Citron et al., 1997; Duff et al., 1996; Borchelt et al.,1996). The identification and cloning of normal as well as mutant PS1and PS2 genes and gene products are described in detail in co-pendingcommonly assigned U.S. application Ser. No. 08/431,048, filed Apr. 28,1995; Ser. No. 08/496,841, filed Jun. 28, 1995; Ser. No. 08/509,359,filed Jul. 31, 1995; and Ser. No. 08/592,541, filed Jan. 26, 1996, thedisclosures of which are incorporated herein by reference.

A new protein which specifically interacts with PS1 and PS2 has recentlybeen discovered. This transmembrane protein, herein referred to as“Presenilin Associated Membrane Protein” or “PAMP”, is expressed inmultiple tissues (e.g., brain, kidney, lung, etc.). PAMP is described inco-pending commonly assigned U.S. application Ser. No. 09/541,094, filedMar. 31, 2000, which is specifically incorporated herein by reference.The PAMP gene and gene product is implicated in the biochemical pathwaysaffected in Alzheimer's Disease (AD), and may also have a role in otherdementias, amyloid angiopathies, and developmental disorders such asspina bifida. Interestingly, the gene associated with inheritedsusceptibility to schizophrenia (see Brzustowicz, supra) also containsthe PAMP gene (Yu et al, 2000).

A need exists for new methods and reagents to more accurately andeffectively diagnose and treat schizophrenia as well as otherneuropsychiatric, neurodevelopmental, and neurodegenerative diseases. Inaddition, further insights into PAMP and its interaction with PSproteins and other components may lead to new diagnostic and treatmentmethods for schizophrenia and other related CNS diseases.

SUMMARY OF THE INVENTION

The present invention provides new uses of the PAMP gene, the product ofthe gene, and mutations and polymorphisms thereof in the study andtreatment of a variety of neurological disorders, especiallyschizophrenia. Applicants have surprisingly discovered that PAMP plays arole in the development of schizophrenia. The PAMP gene and the productof the PAMP gene therefore present new therapeutic targets for thetreatment of a variety of neurological disorders, especiallyschizophrenia. Moreover, the PAMP gene will be useful for generatinganimal and cellular models of schizophrenia.

Thus, PAMP nucleic acids, proteins and peptides, antibodies to PAMP,cells transformed with PAMP nucleic acids, and transgenic animalsaltered with PAMP nucleic acids that possess various utilities, aredescribed herein for the diagnosis, therapy and continued investigationof neuropsychiatric and neurodevelopmental disorders, especiallyschizophrenia. Furthermore, mutant PAMP nucleic acids, proteins, orpeptides, cells transfected with vectors comprising mutant PAMP nucleicacids, transgenic animals expressing mutant PAMP or peptides thereof,and their use in studying neuropsychiatric and neurodevelopmentaldisorders, especially schizophrenia, or developing improved diagnosticor therapeutic methods for such disorders, are presented herein.

The invention provides a method for detecting a mutation in PAMPassociated with neuropsychiatric and neurodevelopmental disorders,especially schizophrenia, comprising obtaining a nucleic acid samplefrom an individual diagnosed with or suspected of having schizophreniaor another neuropsychiatric or neurodevelopmental disorder, andsequencing a gene encoding PAMP from said sample. In particular, suchmethods can identify normal human alleles as well as mutant alleles ofPAMP genes which are causative of or contribute to neuropsychiatric orneurodevelopmental diseases, especially schizophrenia.

The invention also provides a method for diagnosing individualspredisposed to or having a neuropsychiatric and/or neurodevelopmentaldisorder such as schizophrenia, comprising obtaining a nucleic acidsample from an individual diagnosed with or suspected of having such adisorder, and sequencing a gene encoding PAMP from said sample.

The invention also provides a method for diagnosing individualspredisposed to or having a neuropsychiatric and/or neurodevelopmentaldisorder such as schizophrenia, comprising obtaining cells that containnucleic acid encoding PAMP, and under non-pathological conditions,transcribing the nucleic acid, and measuring a level of transcriptionalactivity of the nucleic acid.

The invention further provides a method for diagnosing individualspredisposed to or having a neuropsychiatric or neurodevelopmentaldisorder, especially schizophrenia, comprising obtaining cells from anindividual that express nucleic acid encoding PAMP, and measuring PAMPactivity. Alternatively, PAMP could be isolated from that individual toinvestigate, for example, whether the PAMP amino acid sequence issimilar or different from wild-type PAMP, and/or whether PAMP expressionlevels differ from typical PAMP levels. In an alternative embodiment,the activity or abundance of a PAMP substrate may be measured.

The invention also provides a method for identifying putative agentsthat affect a neuropsychiatric and/or neurodevelopmental disorder,especially schizophrenia, comprising administering one or more putativeagents to a transgenic animal and detecting a change in PAMP activity.

The invention also provides a method for identifying putative agentsthat affect a neuropsychiatric and/or neurodevelopmental disorder,especially schizophrenia, comprising adding one or more said agents tothe reconstituted system described above, and detecting a change in PAMPactivity.

The invention also provides a method for identifying putative agentsthat affect a neuropsychiatric and/or neurodevelopmental disorder,especially schizophrenia, comprising adding one or more said agents tothe complex described above, and detecting a conformational change inPAMP.

The invention also provides a method for identifying proteins thatinteract with PAMP, comprising contacting a substance to thereconstituted system discussed above, and detecting a change in PAMPactivity.

The invention also provides animal and cellular models of schizophreniaor related disorders that comprise a PAMP gene as a therapeutic targetfor the development of drugs which interact with PAMP, and thus may beuseful in the treatment and prevention of schizophrenia or relateddisorders.

Further the invention provides for a method for identifying substancesthat modulate PAMP activity, comprising contacting a sample containingone or more substances with PAMP, or a PAMP mutant, or functionalfragments thereof, and a PAMP substrate, measuring PAMP activity, anddetermining whether a change in PAMP activity occurs. In a preferredembodiment, the substance is a PAMP inhibitor. In another preferredembodiment, the substance stimulates PAMP activity.

These and other aspects of the invention are further elaborated in theDetailed Description of the Invention and Examples, infra.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Predicted amino acid sequences for human (SEQ IDNO:14), mouse (SEQ ID NO:16), D. melanogaster (SEQ ID NO:18) and C.elegans (SEQ ID NO:12) PAMP orthologues.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, the PAMP gene and its product, the PAMPprotein, present new therapeutic targets for the treatment of a varietyof neurological disorders, especially schizophrenia. Provided herein arealso new strategies to create animal and cellular models based on PAMPor PAMP mutants to study schizophrenia and potential treatmentstrategies. The invention also offers the potential for new diagnosticscreening methods for schizophrenia, wherein the PAMP gene and PAMPprotein are investigated.

PAMP

The invention is based, in part, on the discovery that the PAMP gene andthe PAMP protein play critical roles in schizophrenia and otherneuropsychiatric disorders. PAMP (“Presenilin Associated MembraneProtein”), is a novel Type I transmembrane protein that is closelyinvolved in CNS development via its interactions with Notch processing,PS1, PS2 and with the α- and β-secretase derived fragments of βAPP.Multiple studies have indicated that defects in CNS development, such asdefects in neuronal migration, are associated with schizophrenia, andPAMP is linked to this disorder through several lines of evidence, suchas (1) the PAMP gene maps to the same location as the schizophreniasusceptibility gene, as described above; (2) PAMP interacts with PS1,which is associated with migration defects in the central nervous systemin PS1−/− mice (see above); (3) PAMP is involved in the Notch signalingpathway, one gene locus of which (NOTCH4) is implicated in schizophrenia(see above); and (4) a mutation in βAPP is associated with aschizophrenia-like illness (see above). Therefore, PAMP can contributeto the development of schizophrenia via several routes, e.g., throughmutations and/or polymorphisms in PAMP, variations in its expressionlevels, and defects in its interactions with other components in neuraldevelopment and/or migration.

As referred to herein, “PAMP” means a native or mutant full-lengthprotein, or fragments thereof, that interacts with the PAMP-interactingdomain of a presenilin protein. PAMP is also known under the name“Nicastrin”. Human, murine, D. melanogaster and C. elegans orthologuesare provided.

Experimental data indicate that PAMP, PS1, and PS2 exist in the samehigh molecular weight protein complex, and PAMP and PS1 are bothco-localized to intracellular membranes in the endoplasmic reticulum andGolgi apparatus. Abolition of functional expression of a C. eleganshomologue of this protein leads to the development of Notch-likedevelopmental defects. This shows that PAMP is also intimately involvedin the processing of not only βAPP, but also other molecules, such asNotch and its homologues. For example, PAMP can bind to membrane-boundNotch. From expressed sequence tags (EST) databases, it is apparentthat, like PS1 and PS2, PAMP is expressed in multiple tissues.

Various structural features characterize PAMP (GenBank Accession No.Q92542; SEQ ID NO: 14). The nucleotide sequence (SEQ ID NO: 13) of humanPAMP predicts that the gene encodes a Type 1 transmembrane protein of709 amino acids (SEQ ID NO: 14), the protein having a short hydrophilicC-terminus (˜20 residues), a hydrophobic transmembrane domain (15-20residues), and a longer N-terminal hydrophilic domain which containsseveral potentially functional sequence motifs as listed below inTable 1. The PAMP sequence also contains a Trp-Asp (WD) repeat (residue226), at least one “DTG” motif (residues 91-93) present in eukaryoticaspartyl proteases, as well as several “DTA/DTAE” motifs (residues480-482, 504-506) present in viral aspartyl proteases. There are alsofour conserved cysteine residues in the N-terminal hydrophilic domain(Cys₁₉₅, Cys₂₁₃, Cys₂₃₀, and Cys₂₄₈ in human PAMP) having a periodocityof 16-17 residues, which may form a functional domain (e.g., a metalbinding domain or disulfide bridge for tertiary structurestabilization). Subdomains of PAMP have weak homologies to a variety ofpeptidases. For example, residues 322-343, 361-405, and 451-466 have 46%(p=0.03) similarity to another hypothetical protein; C. elegansaminopeptidase hydrolase precursor signal antigen transmembrane receptorzinc glycoprotein (SWISS-PROT; see expasy.ch/sprot on the World-Wide Web(www); Accession No. Q93332). TABLE 1 Potential functional sequencemotifs in PAMP (SEQ ID NO: 14). Potential function PAMP residue (aminoacid sequence) N-asparaginyl glycosylation 45 (NKTA), 55 (NATH), 187(NETK), 200 (NLSQ), 204 (NGSA), 264 (NTTG), 387 (NESV), 417 (NQSQ), 435(NISG), 464 (NVSY), 506 (NFSD), 530 (NNSW), 562 (NTTY), 573 (NLTG), 580(NLTR), 612 (NETD) Glycosaminoglycan attachment 404 (SGAG) Myristolation5 (GGGSGA), 29 (GLCRGN), 61 (GCQSSI), 120 (GLAVSL), 146 (GVYSNS), 167(GNGLAY), 205 (GSAPTF), 294 (GAESAV), 438 (GVVLAD), 446 (GAFHNK), 504(GTNFSD), 576 (GTVVNL) Phosphorylation sites for cAMP- and 232 (RRSS)cGMP-dependent protein kinase Phosphorylation sites for protein 115(TSR), 268 (TLK), 340 (SSR), 384 (SQK), 389 kinase C (SVR), 483 (TAK),614 (TDR), 624 (TAR) Phosphorylation sites for casein kinase 8 (SGAD),280 (TRLD), 361 (SFVE), 372 (TSLE), 455 II (SIYD), 466 (SYPE), 472(SPEE), 641 (SSTE), 647 (TWTE)

The invention is further based on the identification of conservedfunctional domains, based on comparison and evaluation of the predictedamino acid sequences of human (SEQ ID NO: 14), murine (SEQ ID NO: 16),D. melanogaster (SEQ ID NO: 18), and C. elegans (SEQ ID NO: 12)orthologues of PAMP. “PAMP” can be characterized by the presence ofconserved structural features, relative to orthologues from D.melanogaster and C. elegans. Nucleotide sequences encoding homologoushypothetical proteins exist in mice multiple EST, and C. elegans(GenBank; see ncbi.nlm.nih.gov on the World-Wide Web (www); AccessionNo. Z75714; 37% similarity, p=8.7e⁻²⁶) (Wilson et al., 1994). Thesehypothetical murine and nematode proteins have a similar topology andcontain similar functional motifs to human PAMP. The existence of suchhomology predicts that similar proteins will be detected in otherspecies including Xenopus, and Zebra fish, to mention a few suchpossibilities. By comparing the predicted amino acid sequences of human(SEQ ID NO: 14), murine (SEQ ID NO: 16), D. melanogaster (SEQ ID NO:18), and C. elegans (SEQ ID NO: 12) PAMP proteins, we have deduced aseries of conserved functional domains. One domain has chemicalsimilarities to cyclic nucleotide binding domains of other proteins, andmay have some regulatory role on a potential complex formed betweenPS1:PAMP and the C-terminal fragment of βAPP, derived either from α- orβ-secretase. These putative functional domains are sites for therapeutictarget development by deploying drugs which might interact with thesesites to modulate βAPP processing via this complex.

The term “PAMP” also refers to functionally active fragments of theprotein. Such fragments include, but are not limited to, peptides thatcontain an epitope, e.g., as determined by conventional algorithms suchas hydrophilicity/hydrophobicity analysis for antibody epitopes, andamphipathicity or consensus algorithms for T cell epitopes (Spouge etal., 1987; Margalit et al., 1987; Rothbard, 1986; Rothbard and Taylor,1988). More preferably, a functionally active fragment of PAMP is aconserved domain, relative to the D. melanogaster and C. elegansorthologues. A specific functionally active fragment of PAMP is afragment that interacts with PS1 or PS2, or both.

PAMP also encompasses naturally occurring variants, including othermammalian PAMPs (readily identified, as shown herein for murine PAMP,based on the presence of the structural features set forth above),allelic variants of PAMP from other human sources (including variantscontaining polymorphisms that are predictive of disease propensity or ofresponse to pharmacological agents), and mutant forms of PAMP or PAMPgenes that are associated with neurological diseases and disorders (suchas spina bifida), particularly neuropsychiatric disorders (such asschizophrenia). Also included are artificial PAMP mutants created bystandard techniques such as site directed mutagenesis or chemicalsynthesis.

A PAMP “substrate” may be a polypeptide or protein, or any other type ofcompound, with which PAMP interacts physiologically. Examples of PAMPsubstrates include PS1, PS2, and βAPP. Furthermore, A PAMP “ligand” maybe a polypeptide, protein, lipid, carbohydrate, vitamin, mineral, aminoacid, or any other type of compound which binds to PAMP Hypothetically,PAMP may function as a receptor which modulates PS1/PS2/βAPP processingin response to signal (ligand) dependent interactions with PAMP.

Definitions

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, 1989;Glover, 1985; M. J. Gait, 1984; Hames &Higgins, 1985; Hames & S. J.Higgins, 1984; Freshney, 1986; IRL Press, 1986; Perbal, 1984; Ausubel etal., 1994.

If appearing herein, the following terms shall have the definitions setout below.

“Neuropsychiatric disorders” or “diseases” include recognized variantsof overt schizophrenia (e.g., paranoid, catatonic), other relatedpsychoses such as schizoaffective, schizotypal, schizophreniform anddelusional disorders, and personality disorders such as schizoidpersonality disorder, schizotypal personality disorder, and paranoidpersonality disorder (see definitions in DSM-III-R, Diagnostic andStatistical Manual of the American Psychiatric Association; and Flaum etal., 1997).

The use of italics (e.g., PAMP) indicates a nucleic acid molecule (cDNA,mRNA, gene, etc.); normal text (e.g., PAMP) indicates the polypeptide orprotein.

In a specific embodiment, the term “about” or “approximately” meanswithin 20%, preferably within 10%, and more preferably within 5% of agiven value or range. Alternatively, particularly in biological systemswhich are often responsive to order of magnitude changes, the term aboutmeans within an order of magnitude of a given value, preferably within amultiple of about 5-fold, and more preferably within a factor of about2-fold of a given value.

As used herein, the term “isolated” means that the referenced materialis free of components found in the natural environment in which thematerial is normally found. In particular, isolated biological materialis free of cellular components. In the case of nucleic acid molecules,an isolated nucleic acid includes a PCR product, an isolated mRNA, acDNA, or a restriction fragment. In another embodiment, an isolatednucleic acid is preferably excised from the chromosome in which it maybe found, and more preferably is no longer joined to non-regulatory,non-coding regions, or to other genes, located upstream or downstream ofthe gene contained by the isolated nucleic acid molecule when found inthe chromosome. In yet another embodiment, the isolated nucleic acidlacks one or more introns. Isolated nucleic acid molecules can beinserted into plasmids, cosmids, artificial chromosomes, and the like.Thus, in a specific embodiment, a recombinant nucleic acid is anisolated nucleic acid. An isolated protein may be associated with otherproteins or nucleic acids, or both, with which it associates in thecell, or with cellular membranes if it is a membrane-associated protein.An isolated organelle, cell, or tissue is removed from the anatomicalsite in which it is found in an organism. An isolated material may be,but need not be, purified.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate unrelated materials,i.e., contaminants. For example, a purified protein is preferablysubstantially free of other proteins or nucleic acids with which it isassociated in a cell; a purified nucleic acid molecule is preferablysubstantially free of proteins or other unrelated nucleic acid moleculeswith which it can be found within a cell.

As used herein, the term “substantially free” is used operationally, inthe context of analytical testing of the material. Preferably, purifiedmaterial substantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or functional assays, asdescribed infra. A host cell has been “transfected” by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Acell has been “transformed” by exogenous or heterologous DNA when thetransfected DNA is expressed and effects a function or phenotype on thecell in which it is expressed. The term “expression system” means a hostcell transformed by a compatible expression vector and cultured undersuitable conditions e.g. for the expression of a protein coded for byforeign DNA carried by the vector and introduced to the host cell.

Proteins and polypeptides can be made in the host cell by expression ofrecombinant DNA. As used herein, the term “polypeptide” refers to anamino acid-based polymer, which can be encoded by a nucleic acid orprepared synthetically. Polypeptides can be proteins, protein fragments,chimeric proteins, etc. Generally, the term “protein” refers to apolypeptide expressed endogenously in a cell, e.g., the naturallyoccurring form (or forms) of the amino acid-based polymer.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The coding sequences herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of ribonucleicacids or amino acids which comprise all or part of one or more proteins,and may or may not include regulatory DNA sequences, such as promotersequences, which determine for example the conditions under which thegene is expressed.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background.

A coding sequence is “under the control” or “operatively associatedwith” transcriptional and translational control sequences in a cell whenRNA polymerase transcribes the coding sequence into mRNA, which then maybe trans-RNA spliced (if it contains introns) and translated into theprotein encoded by the coding sequence.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence toa host cell, so that the host cell will express the introduced gene orsequence to produce a desired substance, typically a protein or enzymecoded by the introduced gene or sequence. The introduced gene orsequence may also be called a “cloned”, “foreign”, or “heterologous”gene or sequence, and may include regulatory or control sequences usedby a cell's genetic machinery. The gene or sequence may includenonfunctional sequences or sequences with no known function. A host cellthat receives and expresses introduced DNA or RNA has been “transformed”and is a “transformant” or a “clone.” The DNA or RNA introduced to ahost cell can come from any source, including cells of the same genus orspecies as the host cell, or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors include plasmids, phages, viruses, etc. A “cassette”refers to a DNA coding sequence or segment of DNA that codes for anexpression product that can be inserted into a vector at definedrestriction sites. The cassette restriction sites are designed to ensureinsertion of the cassette in the proper reading frame. Generally,foreign DNA is inserted at one or more restriction sites of the vectorDNA, and then is carried by the vector into a host cell along with thetransmissible vector DNA. A segment or sequence of DNA having insertedor added DNA, such as an expression vector, can also be called a “DNAconstruct.” Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes.

A “knockout mammal” is a mammal (e.g., mouse) that contains within itsgenome a specific gene that has been inactivated by the method of genetargeting (see, e.g., U.S. Pat. No. 5,777,195 and U.S. Pat. No.5,616,491). A knockout mammal includes both a heterozygote knockout(i.e., one defective allele and one wild-type allele) and a homozygousmutant. Preparation of a knockout mammal requires first introducing anucleic acid construct that will be used to suppress expression of aparticular gene into an undifferentiated cell type termed an embryonicstem cell. This cell is then injected into a mammalian embryo. Amammalian embryo with an integrated cell is then implanted into a fostermother for the duration of gestation. Zhou, et al., 1995 describes PPCAknock-out mice. Knockout mice can be used to study defects inneurological development or neurodegenerative diseases. Diseasephenotypes that develop can provide a platform for further drugdiscovery.

The term “knockout” refers to partial or complete suppression of theexpression of at least a portion of a protein encoded by an endogenousDNA sequence in a cell. The term “knockout construct” refers to anucleic acid sequence that is designed to decrease or suppressexpression of a protein encoded by endogenous DNA sequences in a cell.The nucleic acid sequence used as the knockout construct is typicallycomprised of (1) DNA from some portion of the gene (exon sequence,intron sequence, and/or promoter sequence) to be suppressed and (2) amarker sequence used to detect the presence of the knockout construct inthe cell. The knockout construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to prevent orinterrupt transcription of the native DNA sequence. Such insertionusually occurs by homologous recombination (i.e., regions of theknockout construct that are homologous to endogenous DNA sequenceshybridize to each other when the knockout construct is inserted into thecell and recombine so that the knockout construct is incorporated intothe corresponding position of the endogenous DNA). The knockoutconstruct nucleic acid sequence may comprise 1) a full or partialsequence of one or more exons and/or introns of the gene to besuppressed, 2) a full or partial promoter sequence of the gene to besuppressed, or 3) combinations thereof. Typically, the knockoutconstruct is inserted into an embryonic stem cell (ES cell) and isintegrated into the ES cell genomic DNA, usually by the process ofhomologous recombination. This ES cell is then injected into, andintegrates with, the developing embryo.

Generally, for homologous recombination, the DNA will be at least about1 kilobase (kb) in length and preferably 3-4 kb in length, therebyproviding sufficient complementary sequence for recombination when theknockout construct is introduced into the genomic DNA of the ES cell.

A “knock-in” mammal is a mammal in which an endogenous gene issubstituted with a heterologous gene or a modified variant of theendogenous gene (Roemer et al., 1991). Preferably, the heterologous geneis “knocked-in” to a locus of interest, for example into a gene that isthe subject of evaluation of expression or function, thereby linking theheterologous gene expression to transcription from the appropriatepromoter (in which case the gene may be a reporter gene; see Elefanty etal., 1998). This can be achieved by homologous recombination, transposon(Westphal and Leder, 1997), using mutant recombination sites (Araki etal., 1997) or PCR (Zhang and Henderson, 1998).

The phrases “disruption of the gene” and “gene disruption” refer toinsertion of a nucleic acid sequence into one region of the native DNAsequence (usually one or more exons) and/or the promoter region of agene so as to decrease or prevent expression of that gene in the cell ascompared to the wild-type or naturally occurring sequence of the gene.By way of example, a nucleic acid construct can be prepared containing aDNA sequence encoding an antibiotic resistance gene which is insertedinto the DNA sequence that is complementary to the DNA sequence(promoter and/or coding region) to be disrupted. When this nucleic acidconstruct is then transfected into a cell, the construct will integrateinto the genomic DNA. Thus, some progeny of the cell will no longerexpress the gene, or will express it at a decreased level, as the DNA isnow disrupted by the antibiotic resistance gene.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is a such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature. In the context of the presentinvention, an gene is heterologous to the recombinant vector DNA inwhich it is inserted for cloning or expression, and it is heterologousto a host cell containing such a vector, in which it is expressed, e.g.,a CHO cell.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g. DNA, or any process, mechanism, or result of such achange. This includes gene mutations, in which the structure (e.g., DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g., protein) expressedby a modified gene or DNA sequence. The term “variant” may also be usedto indicate a modified or altered gene, DNA sequence, enzyme, cell,etc., i.e., any kind of mutant.

“Sequence-conservative variants” of a polynucleotide sequence are thosein which a change of one or more nucleotides in a given codon positionresults in no alteration in the amino acid encoded at that position.

“Function-conservative variants” are those in which a given amino acidresidue in a protein or enzyme has been changed without altering theoverall conformation and function of the polypeptide, including, but notlimited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Amino acids withsimilar properties are well known in the art. For example, arginine,histidine and lysine are hydrophilic-basic amino acids and may beinterchangeable. Similarly, isoleucine, a hydrophobic amino acid, may bereplaced with leucine, methionine or valine. Such changes are expectedto have little or no effect on the apparent molecular weight orisoelectric point of the protein or polypeptide. Amino acids other thanthose indicated as conserved may differ in a protein or enzyme so thatthe percent protein or amino acid sequence similarity between any twoproteins of similar function may vary and may be, for example, from 70%to 99% as determined according to an alignment scheme such as by theCluster Method, wherein similarity is based on the MEGALIGN algorithm. A“function-conservative variant” also includes a polypeptide or enzymewhich has at least 60% amino acid identity as determined by BLAST(Altschul, et al., 1990) or FASTA algorithms, preferably at least 75%,most preferably at least 85%, and even more preferably at least 90%, andwhich has the same or substantially similar properties or functions asthe native or parent protein or enzyme to which it is compared.

An “ortholog” to a protein means a corresponding protein from anotherspecies. Orthologous proteins typically have similar functions indifferent species, and can also be substantially homologous.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987). Such proteins (and their encoding genes) have sequencehomology, as reflected by their sequence similarity, whether in terms ofpercent similarity or the presence of specific residues or motifs. Motifanalysis can be performed using, for example, the program BLOCKS(blocks.fhcrc.org on the World-Wide Web).

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., supra). However, in common usageand in the instant application, the term “homologous,” when modifiedwith an adverb such as “highly,” may refer to sequence similarity andmay or may not relate to a common evolutionary origin.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 80%, and mostpreferably at least about 90 or 95% of the nucleotides match over thedefined length of the DNA sequences, as determined by sequencecomparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Sequencesthat are substantially homologous can be identified by comparing thesequences using standard software available in sequence data banks, orin a Southern hybridization experiment under, for example, stringentconditions as defined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than80% of the amino acids are identical, or greater than about 90% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program, ProteinPredict(dodo.cmpc.columbia.edu/predictprotein on the World-Wide Web), or any ofthe programs described above (BLAST, FASTA, etc.).

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m)(melting temperature) of 55° C., can be used. Moderate stringencyhybridization conditions correspond to a higher T_(m) and highstringency hybridization conditions correspond to the highest T_(m).Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m) for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating T_(m) have been derived (seeSambrook et al., supra, 9.50-9.51). For hybridization with shorternucleic acids, i.e., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra, 11.7-11.8). A minimumlength for a hybridizable nucleic acid is at least about 10 nucleotides;preferably at least about 15 nucleotides; and more preferably the lengthis at least about 20 nucleotides.

The present invention provides antisense nucleic acids (includingribozymes), which may be used to inhibit expression of PAMP, e.g., todisrupt a cellular process (such disruption can be used in an animalmodel or therapeutically). An “antisense nucleic acid” is a singlestranded nucleic acid molecule which, on hybridizing under cytoplasmicconditions with complementary bases in an RNA or DNA molecule, inhibitsthe latter's role. If the RNA is a messenger RNA transcript, theantisense nucleic acid is a counter transcript or mRNA-interferingcomplementary nucleic acid. As presently used, “antisense” broadlyincludes RNA-RNA interactions, RNA-DNA interactions, ribozymes andRNase-H mediated arrest. Antisense nucleic acid molecules can be encodedby a recombinant gene for expression in a cell (e.g., U.S. Pat. No.5,814,500; U.S. Pat. No. 5,811,234), or alternatively they can beprepared synthetically (e.g., U.S. Pat. No. 5,780,607).

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 10, preferably at least 15, and more preferably atleast 20 nucleotides, preferably no more than 100 nucleotides, that ishybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNAmolecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. In one embodiment, a labeled oligonucleotide can be used asa probe to detect the presence of a nucleic acid. In another embodiment,oligonucleotides (one or both of which may be labeled) can be used asPCR primers, e.g., for cloning full length or a fragment of a protein orpolypeptide. In a further embodiment, an oligonucleotide of theinvention can form a triple helix with a nucleic acid (genomic DNA ormRNA) encoding a protein or polypeptide. Generally, oligonucleotides areprepared synthetically, preferably on a nucleic acid synthesizer.Accordingly, oligonucleotides can be prepared with non-naturallyoccurring phosphoester analog bonds, such as thioester bonds, etc.Furthermore, the oligonucleotides herein may also be modified with alabel capable of providing a detectable signal, either directly orindirectly. Exemplary labels include radioisotopes, fluorescentmolecules, biotin, and the like.

Specific non-limiting examples of synthetic oligonucleotides envisionedfor this invention include oligonucleotides that containphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl, or cycloalkl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are those withCH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂and O—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—PO₂—O—CH₂).U.S. Pat. No. 5,677,437 describes heteroaromatic olignucleosidelinkages. Nitrogen linkers or groups containing nitrogen can also beused to prepare oligonucleotide mimics (U.S. Pat. No. 5,792,844 and U.S.Pat. No. 5,783,682). U.S. Pat. No. 5,637,684 describes phosphoramidateand phosphorothioamidate oligomeric compounds. Also envisioned areoligonucleotides having morpholino backbone structures (U.S. Pat. No.5,034,506). In other embodiments, such as the peptide-nucleic acid (PNA)backbone, the phosphodiester backbone of the oligonucleotide may bereplaced with a polyamide backbone, the bases being bound directly orindirectly to the aza nitrogen atoms of the polyamide backbone (Nielsenet al., 1991). Other synthetic oligonucleotides may contain substitutedsugar moieties comprising one of the following at the 2′ position: OH,SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n is from 1 toabout 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF₃; OCF₃; O-; S-, or N-alkyl; O-, S-, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;a fluorescein moiety; an RNA cleaving group; a reporter group; anintercalator; a group for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.Oligonucleotides may also have sugar mimetics such as cyclobutyls orother carbocyclics in place of the pentofuranosyl group. Nucleotideunits having nucleosides other than adenosine, cytidine, guanosine,thymidine and uridine, such as inosine, may be used in anoligonucleotide molecule.

Presenilins

The presenilin genes (PS1-PS1 and PS2-PS2) encode homologous polytopictransmembrane proteins that are expressed at low levels in intracellularmembranes including the nuclear envelope, the endoplasmic reticulum, theGolgi apparatus and some as yet uncharacterized intracytoplasmicvesicles in many different cell types including neuronal andnon-neuronal cells (see U.S. application Ser. No. 08/431,048, filed Apr.28, 1995; Ser. No. 08/496,841, filed Jun. 28, 1995; and Ser. No.08/509,359, filed Jul. 31, 1995; PCT Publication No. WO 96/34099, andU.S. Pat. Nos. 5,986,054, 5,040,540, and 6,020,143, the disclosures ofwhich are specifically incorporated herein by reference; Sherrington etal., 1995; Rogaev et al., 1995; Levy-Lahad et al., 1995; Doan et al.,1996; Walter et al., 1996; De Strooper et al., 1997; Lehmann et al.,1997; Li et al., 1997). Structural studies predict that the presenilinscontain between six and eight transmembrane (TM) domains organized suchthat the N-terminus, the C-terminus, and a large hydrophilic loopfollowing the sixth TM domain are located in the cytoplasm ornucleoplasm, while the hydrophilic loop between TM1 and TM2 is locatedwithin the lumen of membranous intracellular organelles (Doan et al.,1996; De Strooper et al., 1997; Lehmann et al., 1997).

Presenilin Interacting Proteins

Proteins that interact with the presenilins, i.e., PS-interactingproteins, include PAMP, the S5a subunit of the 26S proteasome (GenBank;Accession No. U51007), Rab11 (GenBank; Accession Nos. X56740 andX53143), retinoid X receptor B, also known as nuclear receptorco-regulator or MHC (GenBank Accession Nos. M84820, and X63522), GT24(GenBank Accession No. U81004), β-catenin (Zhou et al., 1997, and Yu etal., supra) as well as armadillo proteins. These and other PS1 bindingproteins are described in Applicants' copending commonly assigned U.S.application Ser. No. 08/888,077, filed Jul. 3, 1997, as well as U.S.application Ser. No. 08/592,541, filed Jan. 26, 1996, and U.S.application Ser. No. 09/541,094, filed Mar. 31, 2000, the disclosures ofwhich are incorporated herein by reference.

PS1 and PS2 interact specifically with at least two members of thearmadillo family of proteins; neuronal plakophilin-related armadilloprotein (Paffenholtz et al., 1997; Paffenholtz et al., 1999; Zhou et al.(2), 1997) and β-catenin, that are expressed in both embryonic andpost-natal tissues. Moreover, the domains of PS1 and PS2 that interactwith these proteins have been identified. Mutations in PS1 and PS2affect the translocation of β-catenin into the nucleus of both nativecells and cells transfected with a mutant PS gene. These interactionsand effects are described in detail in co-pending commonly assigned U.S.application Ser. No. 09/227,725, filed Jan. 8, 1999, the disclosure ofwhich is incorporated herein by reference.

The methods of the present invention are not limited to mutantpresenilins wherein the PAMP-interacting domain is mutated relative tothe wild-type protein. For example, Applicants have observed thatmutations in PS1 (e.g., M146L) outside of the interacting domain (loop)also affect β-catenin translocation. These mutations probably disturbthe presenilin armadillo interactions by altering the function of a highMW complex which contains, e.g., the presenilin and armadillo proteins,as described in Yu et al., 1998. Moreover, a comparison of the human PS1(hPS1) and PS2 (hPS2) sequences reveals that these pathogenic mutationsare in regions of the PS1 protein which are conserved in the PS2protein. Therefore, corresponding mutations in corresponding regions ofPS2 may also be expected to be pathogenic and are useful in the methodsdescribed herein.

PAMP Mutants

Mutant PS1 and PS2 genes, and their corresponding amino acid sequencesare described in Applicants' co-pending U.S. application Ser. No.08/888,077, filed Jul. 3, 1997, and incorporated herein by reference.Examples of PS1 mutations include I143T, M146L, L171P, F177S, A260V,C263R, P264L, P267S, E280A, E280G, A285V, L286V, Δ291-319, L322V, G384A,L392V, C410Y and I439V. Examples of PS2 mutations include N141I, M239Vand I420T.

PAMP mutants may cause biochemical changes similar to those affectingthe onset or progression of schizophrenia. Therefore, artificial PAMPmutations can potentially be used to generate cellular and other modelsystems to design treatments and preventive strategies for schizophreniaand related disorders. Such mutations may also be used for evaluatingwhether PAMP is involved in the pathogenesis of schizophrenia. Since theamyloid-β (Aβ) inducing mutations are found in amino acid residues of asoluble (non-membrane spanning) domain of PAMP, analysis of the normalstructure of this domain and the effects of these and other nearbymutations on the structure of this domain (and the other domains ofPAMP) provide information for the design of specific moleculartherapeutics.

In general, modifications of the sequences encoding the polypeptidesdescribed herein may be readily accomplished by standard techniques suchas chemical syntheses and site-directed mutagenesis. See Gillman et al.,1979; Roberts et al., 1987; and Innis, 1990. Most modifications areevaluated by routine screening via an assay designed to select for thedesired property.

Antibodies to PAMP

According to the invention, PAMP polypeptides produced recombinantly orby chemical synthesis, and fragments or other derivatives or analogsthereof, including fusion proteins and PAMP mutants, may be used as animmunogen to generate antibodies that recognize the PAMP polypeptide.Such antibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments, and an Fab expression library.Such an antibody is preferably specific for human PAMP, PAMP originatingfrom other species, or for post-translationally modified (e.g.phosphorylated, glycosylated) PAMP.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to PAMP polypeptide or derivative or analogthereof. For the production of antibody, various host animals can beimmunized by injection with the PAMP polypeptide, or a derivative (e.g.,fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the PAMPpolypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Antiseramay be collected at a chosen time point after immunization, and purifiedas desired.

For preparation of monoclonal antibodies directed toward the PAMPpolypeptide, or fragment, analog, or derivative thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Köhler and Milstein,1975, as well as the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983; Cote et al., 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., 1985). Production of human antibodies by CDR grafting is describedin U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762 to Queen et al.,and also in U.S. Pat. No. 5,225,539 to Winter and International PatentApplication PCT/WO91/09967 by Adau et al. In an additional embodiment ofthe invention, monoclonal antibodies can be produced in germ-freeanimals (International Patent Publication No. WO 89/12690, published 28Dec. 1989). In fact, according to the invention, techniques developedfor the production of “chimeric antibodies” (Morrison et al., 1984);Neuberger et al., 1984; Takeda et al., 1985) by splicing the genes froma mouse antibody molecule specific for an PAMP polypeptide together withgenes from a human antibody molecule of appropriate biological activitycan be used; such antibodies are within the scope of this invention.Such human or humanized chimeric antibodies are preferred for use intherapy of human diseases or disorders (described infra), since thehuman or humanized antibodies are much less likely than xenogenicantibodies to induce an immune response, in particular an allergicresponse, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778) can be adapted to produce PAMPpolypeptide-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., 1989) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor an PAMP polypeptide, or its derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an PAMP polypeptide, one may assay generatedhybridomas for a product which binds to an PAMP polypeptide fragmentcontaining such epitope. For selection of an antibody specific to anPAMP polypeptide from a particular species of animal, one can select onthe basis of positive binding with PAMP polypeptide expressed by orisolated from cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the PAMP polypeptide, e.g.,for Western blotting, imaging PAMP polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc. using any of thedetection techniques mentioned above or known in the art. Suchantibodies can be used to identify proteins that interact with PAMP, andto detect conformational or structural changes in PAMP.

In a specific embodiment, antibodies that agonize or antagonize theactivity of PAMP polypeptide can be generated. They can also be used toregulate or inhibit PAMP activity intracellular, i.e., the inventioncontemplates an intracellular antibody (intrabody), e.g., single chainFv antibodies (see generally, Chen, 1997; Spitz et al., 1996; Indolfi etal., 1996; Kijima et al., 1995).

PAMP Diagnostic Assays

The nucleotide sequence and the protein sequence and the putativebiological activity of PAMP or PAMP mutants can all be used for thepurposes of diagnosis of individuals who are at-risk for, or whoactually have, a variety of neurodegenerative diseases (includingAlzheimer's disease, Lewy body variant, Parkinson's disease-dementiacomplex, amyotrophic lateral sclerosis etc.), neuropsychiatric diseases(schizophrenia, depression, mild cognitive impairment, benign senescentforgetfulness, age-associated memory loss, etc.), neurodevelopmentaldisorders associated with defects in intracellular signal transductionmediated by factors such as Notch, Delta, Wingless, etc., and neoplasms(e.g. bowel cancer, etc.) associated with abnormalities of PS1/PAMP/PS2mediated regulation of cell death pathways. These diagnostic entitiescan be used by searching for alterations in: the nucleotide sequence ofPAMP; in the transcriptional activity of PAMP; in the protein level asmonitored by immunological means (e.g., ELISA and Western blots); in theamino acid sequence (as ascertained by Western blotting, amino acidsequence analysis, mass spectroscopy); and/or in the biological activityof the PAMP protein as measured by either in vivo methods (e.g.,monitoring βAPP processing and the production of amyloid-β peptide (Aβ),or other suitable protein substrates for PAMP including Notch, etc.), orby in vitro assays (using either whole cell or cell-free assays tomeasure processing of suitable substrates including βAPP or partsthereof, and other proteins such as Notch). Any of these assays can alsobe performed in a transgenic animal model as well, e.g., to measure theeffect of a drug or a mutation or overexpression of a different gene invivo.

PAMP Screening Assays

Identification and isolation of PAMP provides for development ofscreening assays, particularly for high throughput screening ofmolecules that up- or down-regulate the activity of PAMP, e.g., bypermitting expression of PAMP in quantities greater than can be isolatedfrom natural sources, or in indicator cells that are speciallyengineered to indicate the activity of PAMP expressed after transfectionor transformation of the cells. Any screening technique known in the artcan be used to screen for PAMP agonists or antagonists. The presentinvention contemplates screens for small molecule ligands or ligandanalogs and mimics, as well as screens for natural ligands that bind toand agonize or antagonize the activity of PAMP in vivo. For example,natural products libraries can be screened using assays of the inventionfor molecules that agonize or antagonize PAMP activity.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, 1990; Cwirla, etal., 1990; Devlin et al., 1990), very large libraries can be constructed(10⁶-10⁸ chemical entities). A second approach uses primarily chemicalmethods, of which the Geysen method (Geysen et al., 1986; Geysen et al.,1987; and the method of Fodor et al. (1991) are examples. Furka et al.,1988, Furka, 1991, Houghton (U.S. Pat. No. 4,631,211) and Rutter et al.(U.S. Pat. No. 5,010,175) describe methods to produce a mixture ofpeptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., 1993; Ohlmeyeret al., 1993; Lam et al., WO 92/00252; Kocis et al., WO 9428028) and thelike can be used to screen for PAMP ligands according to the presentinvention.

Knowledge of the primary sequence of the protein, and the similarity ofthat sequence with proteins of known function, can provide an initialclue as to the inhibitors or antagonists of the protein. As noted above,identification and screening of antagonists is further facilitated bydetermining structural features of the protein, e.g., using X-raycrystallography, neutron diffraction, nuclear magnetic resonancespectrometry, and other techniques for structure determination. Thesetechniques provide for the rational design or identification of agonistsand antagonists.

The PAMP protein sequence (including parts thereof) can be used todeduce the structural organization and topology of PAMP through the useof a variety of techniques including CD spectroscopy, nuclear magneticresonance (NMR) spectroscopy, X-ray crystallography, and molecularmodeling. Sequences for PAMP or PAMP mutants can also be used toidentify proteins which interact with PAMP either in concert with PS1and PS2, or independently, using a variety of methods includingco-immunoprecipitation, yeast two hybrid interaction trap assays, yeastthree hybrid interaction trap assays, chemical cross-linking andco-precipitation studies, etc. These and other methods are describedmore fully in co-pending and commonly assigned U.S. application Ser. No.08/888,077, filed Jul. 3, 1997, and Ser. No. 09/227,725, filed Jan. 8,1999, both of which are incorporated herein by reference. Identificationof these interacting partners will then lead to their use to furtherdelineate the biochemical pathways leading to the above-mentioneddiseases.

Finally, the structural analysis of PAMP, when combined with structuralanalysis of PS1 and PS2, and other proteins which interact with PAMP orPAMP mutants, will identify the structural domains that mediateinteractions between these molecules and which also confer biologicalactivity on PAMP (or PAMP and these other molecules). These structuraldomains, and other functional domains, which can modulate the activityof these structural domains, can all be modified through a variety ofmeans, including but not limited to site-directed mutagenesis, in orderto either augment or reduce the biological activity. The structure andtopology of these domains can all be used as a basis for the rationaldesign of pharmaceuticals (small molecule conventional drugs or novelcarbohydrate, lipid, DNA/RNA or protein-based high molecular weightbiological compounds) to modulate (increase or decrease) the activity ofPAMP and/or the PAMP PS1/PS2 complex, and/or the activity of thePAMP/other protein complexes. For example, using structural predictioncalculations, possibly in conjunction with spectroscopic data likenuclear magnetic resonance, circular dichroism, and otherphysical-chemical structural data, or crystallographic data, or both,one can generate molecular models for the structure of PAMP. Thesemodels, in turn, are important for rational drug design. Drug candidatesgenerated using a rational drug design program can then be applied forthe treatment and/or prevention of the above-mentioned diseases, and canbe administered through a variety of means including: as conventionalsmall molecules through enteral or parenteral routes; via inclusion inliposome vehicles; through infusion in pumps inserted into variousorgans (e.g., via Omaya pumps inserted into the cerebral ventricles);via the transplantation of genetically-modified cells expressingrecombinant genes; or via the use of biological vectors (e.g.,retrovirus, adenovirus, adeno-associated virus, Lentivirus, or herpessimplex virus-based vectors) which allow targeted expression ofappropriately modified gene products in selected cell types. It shouldbe noted that the recombinant proteins described above may be thewild-type PAMP, a genetically-modified PAMP, a wild-type PS1/PS2, agenetically-modified PS1/PS2, or a specially-designed protein or peptidewhich is designed to interact with either the functional domains of PAMP(or the PAMP/PS1/PS2/other protein complex) or to interact with thedomains which modulate the activity of the functional domains of PAMP.

PAMP In Vitro and In Vivo Models

The PAMP nucleotide sequence can be used to make cell-free systems,transfected cell lines, and animal models (invertebrate or vertebrate)of the neurodegenerative and other diseases outlined above. These animaland cell models may involve over-expression of all or part of PAMP orPAMP mutants, e.g., as mini-gene cDNA transgene constructs under theregulation of suitable promoter elements carried in vectors such ascos-Tet for transgenic mice and pcDNA (Invitrogen, California) intransfected cell lines. Animal and cellular models can also be generatedby via homologous recombination mediated targeting of the endogenousgene to create artificially mutant sequences (knock-in gene targeting);or loss of function mutations (knock-out gene targeting); bytranslocation of P-elements; and by chemical mutagenesis. Animal,cellular and cell-free model systems can be used for a variety ofpurposes including the discovery of diagnostics and therapeutics forthis disease.

Included within the scope of this invention is a mammal in which two ormore genes have been knocked out or knocked in, or both. Such mammalscan be generated by repeating the procedures set forth herein forgenerating each knockout construct, or by breeding to mammals, each witha single gene knocked out, to each other, and screening for those withthe double knockout genotype.

Regulated knockout animals can be prepared using various systems, suchas the tet-repressor system (see U.S. Pat. No. 5,654,168) or the Cre-Loxsystem (see U.S. Pat. No. 4,959,317 and U.S. Pat. No. 5,801,030).

Transgenic mammals can be prepared for evaluating the molecularmechanisms of PAMP, and particularly human PAMP function. Such mammalsprovide excellent models for screening or testing drug candidates. It ispossible to evaluate compounds or diseases on “knockout” animals, e.g.,to identify a compound that can compensate for a defect in PAMPactivity. Alternatively, PAMP (or mutant PAMP), alone or in combinationwith βAPP, PS1, PS2, and/or Notch, or some other component (double ortriple transgenics) “knock-in” mammals can be prepared for evaluatingthe molecular biology of this system in greater detail than is possiblewith human subjects. Both technologies permit manipulation of singleunits of genetic information in their natural position in a cell genomeand to examine the results of that manipulation in the background of aterminally differentiated organism. These animals can be evaluated forlevels of mRNA or protein expression, processing of βAPP, or developmentof a condition indicative of inappropriate gene expression, e.g., Notchphenotype or another phenotype as set forth above, or neurodegeneration,including cognitive deficits, learning or memory deficits, orneuromuscular deficits.

Various transgenic animal systems have been developed. Mice, rats,hamsters, and other rodents are popular, particularly for drug testing,because large numbers of transgenic animals can be bred economically andrapidly. Larger animals, including sheep, goats, pigs, and cows, havebeen made transgenic as well. Transgenic D. melanogaster and C. eleganscan also be made and, using known genetic methods, may offer the abilityto identify upstream and downstream modifiers of a PAMP phenotype.Transgenic animals can also be prepared by introducing the transgene ona vector; such animals, which are not modified in the germ line and areonly transiently transgenic, naturally cannot pass along the geneticinformation to their progeny.

In another series of embodiments, transgenic animals are created inwhich (i) a human PAMP, or a mutant human PAMP, is stably inserted intothe genome of the transgenic animal; and/or (ii) the endogenous PAMPgenes are inactivated and replaced with their human counterparts. See,e.g., Coffman, 1997; Esther et al., 1996; Murakami et al., 1996. Suchanimals can be treated with candidate compounds and monitored for theeffects of such drugs on PAMP cavity.

PAMP Gene Therapy

As discussed above, abnormalities in PAMP expression and/or interactionswith PS1/PS2/βAPP are associated with severe neurological deficits.Thus, the present invention provides for treatment of such deficitseither with a drug discovered using a screening assay or transgenicanimal model, or both, as set forth above, or by replacing a defectivePAMP gene with a functional gene by gene therapy.

A gene encoding PAMP, a PAMP mutant, or alternatively a negativeregulator of PAMP such as an antisense nucleic acid, intracellularantibody (intrabody), or dominant negative PAMP (which may betruncated), can be introduced in vivo, ex vivo, or in vitro using aviral or a non-viral vector, e.g., as discussed above. Expression intargeted tissues can be effected by targeting the transgenic vector tospecific cells, such as with a viral vector or a receptor ligand, or byusing a tissue-specific promoter, or both. Targeted gene delivery isdescribed in WO 95/28494, published October 1995.

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin 12 (IL-12), interferon-γ (IFNγ), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors (see, e.g., Wilson, 1995). In that regard, it isadvantageous to employ a viral vector that is engineered to express aminimal number of antigens.

Herpes virus vectors. Because herpes virus is trophic for cells of thenervous system (neural cells), it is an attractive vector for deliveryof function PAMP genes. Various defective (non-replicating, and thusnon-infectious) herpes virus vectors have been described, such as adefective herpes virus 1 (HSV1) vector (Kaplitt et al., 1991;International Patent Publication No. WO 94/21807, published Sep. 29,1994; International Patent Publication No. WO 92/05263, published Apr.2, 1994).

Adenovirus vectors. Adenoviruses are eukaryotic DNA viruses that can bemodified to efficiently deliver a nucleic acid of the invention to avariety of cell types in vivo, and has been used extensively in genetherapy protocols, including for targeting genes to neural cells.Various serotypes of adenovirus exist. Of these serotypes, preference isgiven to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) oradenoviruses of animal origin (see WO94/26914). Those adenoviruses ofanimal origin which can be used within the scope of the presentinvention include adenoviruses of canine, bovine, murine (example: Mav1,Beard et al., 1990), ovine, porcine, avian, and simian (example: SAV)origin. Preferably, the adenovirus of animal origin is a canineadenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan or A26/61strain (ATCC VR-800), for example). Various replication defectiveadenovirus and minimum adenovirus vectors have been described for genetherapy (WO94/26914, WO95/02697, WO94/28938, WO94/28152, WO94/12649,WO95/02697 WO96/22378). The replication defective recombinantadenoviruses according to the invention can be prepared by any techniqueknown to the person skilled in the art (Levrero et al., 1991; EP 185573; Graham, 1984; Graham et al., 1977). Recombinant adenoviruses arerecovered and purified using standard molecular biological techniques,which are well known to one of ordinary skill in the art.

Adeno-associated viruses. The adeno-associated viruses (AAV) are DNAviruses of relatively small size which can integrate, in a stable andsite-specific manner, into the genome of the cells which they infect.They are able to infect a wide spectrum of cells without inducing anyeffects on cellular growth, morphology or differentiation, and they donot appear to be involved in human pathologies. The AAV genome has beencloned, sequenced and characterized. The use of vectors derived from theAAVs for transferring genes in vitro and in vivo has been described (seeWO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No.5,139,941, EP 488 528). The replication defective recombinant AAVsaccording to the invention can be prepared by co-transfecting a plasmidcontaining the nucleic acid sequence of interest flanked by two AAVinverted terminal repeat (ITR) regions, and a plasmid carrying the AAVencapsidation genes (rep and cap genes), into a cell line which isinfected with a human helper virus (for example an adenovirus). The AAVrecombinants which are produced are then purified by standardtechniques.

Retrovirus vectors. In another embodiment the gene can be introduced ina retroviral vector, e.g., as described in Anderson et al., U.S. Pat.No. 5,399,346; Mann et al., 1983; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., 1988; Temin etal., U.S. Pat. No. 5,124,263; EP 453242, EP178220; Bernstein et al.,1985; McCormick, 1985; International Patent Publication No. WO 95/07358,published Mar. 16, 1995, by Dougherty et al.; and Kuo et al., 1993. Theretroviruses are integrating viruses which infect dividing cells. Theretrovirus genome includes two LTRs, an encapsidation sequence and threecoding regions (gag, pol and env). In recombinant retroviral vectors,the gag, pol and env genes are generally deleted, in whole or in part,and replaced with a heterologous nucleic acid sequence of interest.These vectors can be constructed from different types of retrovirus,such as MoMuLV (“murine Moloney leukemia virus”), MEV (“murine Moloneysarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosisvirus”); RSV (“Rous sarcoma virus”) and Friend virus. Suitable packagingcell lines have been described in the prior art, in particular the cellline PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO90/02806) and the GP+envAm-12 cell line (WO 89/07150). In addition, therecombinant retroviral vectors can contain modifications within the LTRsfor suppressing transcriptional activity as well as extensiveencapsidation sequences which may include a part of the gag gene (Benderet al., 1987). Recombinant retroviral vectors are purified by standardtechniques known to those having ordinary skill in the art.

Retrovirus vectors can also be introduced by recombinant DNA viruses,which permits one cycle of retroviral replication and amplifiestransfection efficiency (see WO 95/22617, WO 95/26411, WO 96/39036, WO97/19182).

Lentivirus vectors. In another embodiment, lentiviral vectors are can beused as agents for the direct delivery and sustained expression of atransgene in several tissue types, including brain, retina, muscle,liver and blood. The vectors can efficiently transduce dividing andnon-dividing cells in these tissues, and maintain long-term expressionof the gene of interest. For a review, see, Naldini, 1998; see alsoZufferey, et al., 1998). Lentiviral packaging cell lines are availableand known generally in the art. They facilitate the production ofhigh-titer lentivirus vectors for gene therapy. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linewhich can generate virus particles at titers greater than 106 IU/ml forat least 3 to 4 days (Kafri, et al., 1999). The vector produced by theinducible cell line can be concentrated as needed for efficientlytransducing nondividing cells in vitro and in vivo.

Non-viral vectors. A vector can be introduced in vivo in a non-viralvector, e.g., by lipofection, with other transfection facilitatingagents (peptides, polymers, etc.), or as naked DNA. Synthetic cationiclipids can be used to prepare liposomes for in vivo transfection, withtargeting in some instances (Felgner, et. al., 1987; Felgner andRingold, 1989; see Mackey, et al., 1988; Ulmer et al., 1993). Usefullipid compounds and compositions for transfer of nucleic acids aredescribed in International Patent Publications WO95/18863 andWO96/17823, and in U.S. Pat. No. 5,459,127. Other molecules are alsouseful for facilitating transfection of a nucleic acid in vivo, such asa cationic oligopeptide (e.g., International Patent PublicationWO95/21931), peptides derived from DNA binding proteins (e.g.,International Patent Publication WO96/25508), or a cationic polymer(e.g., International Patent Publication WO95/21931). Recently, arelatively low voltage, high efficiency in vivo DNA transfer technique,termed electrotransfer, has been described (Mir et al., 1998; WO99/01157; WO 99/01158; WO 99/01175). DNA vectors for gene therapy can beintroduced into the desired host cells by methods known in the art,e.g., electroporation, microinjection, cell fusion, DEAE dextran,calcium phosphate precipitation, use of a gene gun (ballistictransfection), or use of a DNA vector transporter (see, e.g., Wu et al.,1992; Wu and Wu, 1988; Hartmut et al., Canadian Patent ApplicationNo.2,012,311, filed Mar. 15, 1990; Williams et al., 1991).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992; Wu and Wu, 1987). U.S. Pat. Nos. 5,580,859 and 5,589,466disclose delivery of exogenous DNA sequences, free of transfectionfacilitating agents, in a mammal.

EXAMPLES

The present invention will be further understood by reference to thefollowing examples, which are provided as exemplary of the invention andnot by way of limitation.

Example 1 A Novel PAMP that Mediates βAPP Processing and Notch/Glp1Signal Transduction

This example shows that both PS1 and PS2 interact with a novel Type Itransmembrane protein, PAMP, and that this novel protein also interactswith α- and β-secretase derived fragments of βAPP. We also show thatabolition of functional expression of the C. elegans homologue of theprotein leads to a developmental phenotype (anterior pharynx aph-2)which is thought to be due to inhibition of the glp/Notch signalingpathway. This novel protein is therefore positioned to mediate both thegain of function and loss of function phenotypes associated withpresenilin missense mutations and presenilin knockouts, respectively.

Materials and Methods

Antibodies against PS1, PS2 and βAPP. An antibody, termed 1142, directedagainst PS1, was raised to a peptide segment corresponding to residues30-45 of PS1 (Levesque et al., 1998; Yu et al., 1998). The peptide wassynthesized by solid-phase techniques and purified by reverse phase highpressure liquid chromatography (HPLC). Peptide antigens were linked tokeyhole limpet hemocyanin (KLH) and used, in combination with completeFreud's adjuvant, to innoculate New Zealand White rabbits. Antisera fromthree rabbits was pooled, ammonium precipitated and the antibody waspurified with Sulfo-link (Pierce) agarose-peptide affinity columns.Other antibodies used include antibody 369, a polyclonalrabbit-anti-human antibody directed against the C-terminus of human βAPP(Buxbaum et al., 1990); antibody 14 (Ab14), a rabbit polyclonal antibodyraised against residues 1-25 of human PS1 (Seeger et al., 1997);antibody α-PS1-CTF, a polyclonal rabbit antibody directed against thePS1 loop; and antibody DT2, a monoclonal antibody raised to a GST-fusionprotein containing the PS2 N-terminal sequence from residues 1-87.

Preparation of presenilin associated components. To identify membraneassociated components of the presenilin complex, an immunoaffinityprocedure was used to extract PS1 and tightly associated membraneproteins from semi-purified intracellular membrane fractions. Humanembryonic kidney cells (HEK) 293 (ATCC) with a stable over-expression ofmoderate level wild type human PS1, were grown to confluence, washedtwice with ice-cold phosphate-buffered saline, and then homogenized withBuffer A (0.25 M sucrose, 20 mM HEPES pH 7.2, 2 mM EGTA, 2 mM EDTA, 1 mMDTT, and a protease inhibitor cocktail containing 5 mg/ml each ofLeupeptin, Antipain, pepstatin A, Chymostatin, E64, Aprotinin, and 60mg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF)). The cellhomogenates were centrifuged 1000×g for 10 minutes to remove celldebris. The supernatant was then centrifuged 10,000×g for 60 minutes.The resulting membrane pellet was resuspended in Buffer B (20 mM HEPESpH 7.2, 1 M KCl, 2 mM EGTA, 2 mM EDTA, 1 mM DTT, and protease inhibitorcocktail as above) and incubated for 45 minutes with agitation at 4° C.Cell membranes were collected again by centrifugation at 107,000×g for60 minutes. The cell membranes were then lysed on ice for 60 minuteswith Buffer C (1% Digitonin, 20 mM HEPES pH 7.2, 100 mM KCl, 2 mM EGTA,2 mM EDTA, 1 mM DTT, and protease inhibitors cocktail). After spinning10,000×g for 15 minutes, the protein extract was adjusted with Buffer Cto contain 5 mg/ml protein. A total of 0.5 g of protein was obtained.

Isolation. The extracted proteins were then subjected to fractionationwith 10-40% glycerol gradient containing 0.5% Digitonin as described (Yuet al., 1998). After being verified by Western blotting with anti-PS1antibodies, the peak fractions containing PS1 were pooled and incubatedovernight with Protein A/G agarose coupled with either antibody 1142 ora control IgG purified from preimmune rabbit serum. The Protein A/Gagarose beads were washed three times with Buffer D (1% Digitonin, 20 mMHEPES pH 7.2, 100 mM KCl, protease inhibitors cocktail), and three timeswith Buffer E (0.5% Digitonin, 0.5% CHAPS, 20 mM HEPES pH 7.2, 100 mMKCl, 10 mM CaCl₂, 5 mM MgCl₂, and the protease inhibitor cocktail asabove). Isolated protein complexes were eluted from the beads with 0.1MGlycine-HCl, pH 3.0, and then neutralized with 1M Tris. Proteins werethen separated by Tris-Glycine SDS-PAGE gels and stained with silverstain and Coomassie Blue stain. The staining of the immuno-purifiedproteins displayed two intense bands in addition to those of thepresenilin holoprotein and its fragments.

Sequence analysis. Individual protein bands were cut out and analyzedwith solid-phase extraction capillary electrophoresis massspectrometry/mass spectrometry (SPE-CE-MS/MS). Briefly, protein bandswere first digested in-gel with trypsin; the digested proteins wereextracted and dried in a speed vacuum down to concentrate the peptides;and the peptides thereafter separated with micro LC and analyzed byon-line tandem mass spectrometry (Figeys et al., 1999). Nucleotide andamino acid sequence homology searches were conducted using the BLASTalgorithm, and motif analyses performed using the program BLOCKS.

General transfection and analysis methods. Based on the human PAMPsequence, public databases (e.g., GenBank; see ncbi.nlm.nih.gov on theWorld-Wide Web (www)) were searched for homologous ESTs (SEQ IDNOs:3-10), which were collected into a few contigs. These contigs allturned out to be correct, but did not cover full-length mouse and D.melanogaster cDNAs.

Full length murine (SEQ ID NO: 15), human (SEQ ID NO: 13) and D.melanogaster (SEQ ID NO: 17) PAMP cDNAs were obtained usingoligonucleotides designed from partial cDNA/EST sequences in publicdatabases to screen appropriate cDNA libraries, for 5′RACE, and/or forRT-PCR experiments. A PAMP expression construct was generated byinserting human PAMP cDNA in-frame with the V5 epitope of pcDNA6(Invitrogen) at the C-terminus of PAMP. HEK293 cells with a stableexpression of PS1/PS2 and βAPP_(sw) were transiently transfected witheither V5-tagged PAMP or empty plasmid (mock transfection control).Duplicate experiments were performed by: (1) transient transfection ofV5-PAMP and βAPP₆₉₅ (or empty vector plus βAPP₆₉₅ as a mock transfectioncontrol) into murine embryonic fibroblasts stably infected with humanPS1 expressed from a retroviral vector construct (Clontech, CA); or (2)transient transfection of V5-PAMP (or an empty plasmid) into HEK293 celllines with a stable expression of the C-terminal 99 amino acids of βAPPwith a preceding artificial signal peptide (spC100-APP) together witheither wild type PS1, PS1-L392V, or PS1-D385A. Cells were lysed with aDigitonin lysis buffer or with 1% NP40, and the protein extracts weresubjected to gradient fraction, immunoprecipitation or direct Westernblotting as described (Yu et al., 1998). PS1 was immunodetected orimmunoprecipitated with antibodies 14 or a-PS1-CTF; and PS2 wasimmunodetected or immunoprecipitated with antibody DT2. FL-βAPP and itsC-terminal α- and β-secretase derivatives were detected using antibody369.

Results

Isolation of PAMP. Immunoprecipitation of PS1 protein complexes,followed by SDS-PAGE with Coomassie Blue and silver staining, yieldedtwo intense bands in addition to presenilin holoprotein. These bandswere characterized by mass spectroscopy analysis. Mass spectroscopyanalysis revealed several armadillo repeat containing peptides,(previously known to functionally interact with presenilins (Yu et al.,1998; Zhou et al. (1), 1997; Nishimura M, et al., 1999), and a novelpeptide (PAMP) which had a sequence identified to that predicted for ananonymous, partial cDNA (Genbank; Accession No. D87442). The cDNAsequence predicted an open reading frame of 709 amino acids (SEQ ID NO:14), which contains a putative N-terminal signal peptide, a longN-terminal hydrophilic domain with sequence motifs for glycosylation,N-myristoylation and phosphorylation, a ˜20 residue hydrophobic putativetransmembrane domain, and a short hydrophilic C-terminus of 20 residues(FIGS. 1A and 1B).

Orthologous PAMP proteins. The PAMP amino acid sequence had nosignificant homology to other proteins within available databases,except for a hypothetical C. elegans protein (www.ncbi.nih.gov;Accession No. Q23316) (p=2×10⁻²⁸; identity=22%; similarity=39%) (SEQ IDNO: 12) ascertained from a genomic DNA sequence (FIGS. 1A and 1B). Inaddition to strong primary amino acid sequence conservation, this C.elegans protein has a very similar topology to human PAMP, suggestingthat it is the nematode orthologue of human PAMP.

In the absence of functional clues arising from homologies to otherknown proteins, the predicted amino acid sequences of the murine (SEQ IDNO: 16) and D. melanogaster (SEQ ID NO: 18) orthologues of PAMP werecloned and examined. The four orthologous PAMP proteins had a similartopology and significant sequence conservation near residues 306-360,419-458, and 625-662 of human PAMP (SEQ ID NO: 14) (FIGS. 1A and 1B).Motif analysis of these conserved domains revealed a weak similarity(strength=1046) between residues 625-641 (ARLARALSPAFELSQWS; SEQ ID NO:19) of mouse and human PAMP to cyclic nucleotide binding domains. Whilethe putative transmembrane domain sequences were not highly conserved,all four orthologues contained a conserved serine residue within thishydrophobic domain. Finally, there were four conserved cysteine residuesin the—terminal hydrophilic domain (Cys₁₉₅, Cys₂₁₃, Cys₂₃₀, and Cys₂₄₈in human PAMP) which had a periodicity of 16-17 residues in theN-terminus, and may form a functional domain (e.g., a metal bindingdomain or disulfide bridges for stabilizing the tertiary structure ofPAMP/PAMP complexes).

Interaction of PAMP with presenilin 1. To confirm the authenticity ofthe PAMP:PS1 interaction, HEK293 cells were transiently transfected withPAMP cDNA (SEQ ID NO: 13) tagged at the 3′-end with a V5-epitope encodedfrom the pcDNA6 vector. The conditioned media were collected 20 hr aftertransient transfection with PAMP (or with empty vector), and the Aβ₄₀and Aβ₄₂ levels were measured by ELISA (Zhang L, et al., 1999). InWestern blots of lysates of these cells, the use of anti-V5 (Invitrogen,CA) and enhanced chemiluminescence (Amersham) detected aV5-immunoreactive band of ˜110 kDa which was reduced to ˜80 kDafollowing Endo H digestion (equivalent to the size predicted from thePAMP amino acid sequence), confirming the predicted glycosylation ofPAMP. In addition, a series of about 7-10 kDa fragments were observed,which are predicted to contain the TM domain and short C-terminus ofPAMP plus the 3 kDa V5-epitope. These C-terminal derivatives may beauthentic cleavage products of full-length PAMP, or, alternatively, aproteolytic artifact arising from the attachment of the V5-epitope tothe C-terminus of PAMP.

Reciprocal immunoprecipitation studies in cells expressing combinationsof transfected V5-tagged-PAMP, transfected wild type or mutant PS1,transfected wild type PS2, or endogenous presenilins, confirmed thePS1:PAMP interaction, and showed a similar interaction between PAMP andPS2. In contrast, immunoprecipitation of other ER-resident proteins(e.g., calnexin) failed to show any evidence of an interaction betweenthese proteins and PAMP. Glycerol velocity gradient analysis of thenative conformation of PAMP revealed that PAMP was co-eluted into thesame high molecular weight fractions as PS1 and PS2, indicating that itis an authentic component of the high molecular weight presenilinprotein complexes. These biochemical data were supported byimmunocytochemical studies, which showed that transfected PAMP andendogenous PS1 strongly co-localized in the ER and Golgi in MDCK caninekidney/epithelial cells (ATCC). Similar studies with PS2 confirmed thatPAMP also tightly associates with both endogenous PS2 in human brain andwith transfected PS2 in HEK293 cells.

The PAMP gene. Chromosomal locations and genetic map positions of themurine and human PAMPS were obtained from public genetic andtranscriptional maps (www.ncbi.nlm.nih.gov). The gene encoding PAMP islocated on human chromosome 1 near the genetic markers D1S1595 andD1S2844. The 5′-end of the PAMP gene is embedded in the 5′-end of thecoatmer gene encoded on the opposite strand. The human PAMP gene isclose to a cluster of markers which have yielded positive, butsub-significant evidence for linkage to or association with AlzheimerDisease in two independent genome wide surveys (Kehoe et al., 1999). Themurine PAMP maps within a 700 Kb interval of murine chromosome 1 whichcontains the gene defect associated with Looptail phenotype in mice(Underhill et al., 1999). Mice heterozygous for Looptail showdevelopmental defects in dorsal axial structures including notochord,brain, spinal cord, and somites (Greene et al., 1998), which arereminiscent of those observed in PS1^(−/−) mice (Shen J, et al., 1997;Wong et al., 1997). These observations suggest that the presenilin: PAMPcomplex may be involved in both βAPP and Notch processing.

C. elegans homolog of PAMP. The C. elegans homolog of PAMP correspondsto the aph-2 gene. Mutations in aph-2 have been identified in a screenfor mutants with phenotypes identical to embryonic mutant phenotypescaused by loss of glp-1 activity, i.e., lack of an anterior pharynx,e.g. cDNA clone. The EST corresponding to aph-2, (cDNA clone yk477b8,kindly provided by Y. Kohara, National Institute of Genetics, Japan) wassequenced and the coding region (SEQ ID NO: 11) found to match exactlythe Genefinder prediction made by the C. elegans sequencing consortium(Genbank; Accession No. Z75714). Double stranded RNA interference (RNAi)confirmed the mutant phenotype of aph-2. Sense and antisense RNA weretranscribed in vitro from PCR product amplified from the phage yk477b8.After annealing equal quantities of sense and antisense products, thedsRNA product made was injected into L4 stage wild-type worms. Thechosen line of worms, designated lin-12(n302) (Greenwald and Seydoux,1990; Greenwald, et al., 1983) was obtained from the CaenorhabditisGenetics Center. Injected animals were transferred to fresh plates dailyand the progeny scored at least 36 hours after injection for theembryonic lethal phenotype and 4-5 days after injection for theegg-laying phenotypes. Animals injected with dsRNA from yk477b8 templateproduced eggs that lacked an anterior pharynx. These results support thenotion that aph-2/PAMP contributes to cell interactions mediated byglp-1/Notch in the embryo.

Functional role for the PAMP:presenilin complexes in βAPP processing. Toexamine a functional role for the PAMP:presenilin complexes in βAPPprocessing, the interactions between PAMP, PS1, and βAPP, and itsderivatives were investigated. The cell lines used were transientlytransfected with V5-tagged PAMP, and stably expressing wild type βAPP₆₉₅in addition to wild type PS1, wild type PS2, PS1-L392V mutant, orPS1-D385A mutant. The PS1-L392V mutation is a pathogenic mutationassociated with familial AD (Sherrington et al., 1995) and withincreased secretion of Aβ₄₂ (Scheuner et al., 1996; Citron et al.,1997). The PS1-D385A mutation is a loss of function mutation associatedwith inhibition of PS1 endoproteolysis and a decrease in γ-secretaseactivity (Wolfe et al., 1999). The conditioned media were collected 20hr after transient transfection with PAMP (or with empty vector), andthe Aβ₄₀ and Aβ₄₂ levels were measured by ELISA (Zhang et al., 1999).Analysis of Western blots from these co-immunoprecipitation experimentsrevealed that PAMP holoprotein (and C-terminally tagged proteolyticfragments of PAMP) interacted in equivalent degrees with wild type PS1,wild type PS2, PS1-L392V mutant, and PS1-D385A mutant proteins. Inaddition, PAMP holoprotein and the C-terminal proteolytic fragments ofPAMP also co-immunoprecipitated with the C-terminal proteolyticfragments of βAPP but not βAPP holoprotein in lysates of cellsexpressing either βAPP holoprotein or just the C-terminal 99 amino acidsof βAPP. Significantly, compared to cells expressing equivalentquantities of wild type PS1, cell lines expressing pathogenic mutationsof PS1 showed increased amounts of C-terminal βAPP fragmentsco-immunoprecipitating with PAMP. Conversely, cell lines expressing theloss-of-function PS1-D385A mutation showed greatly reduced amounts ofC-terminal βAPP derivatives co-immunoprecipitating with PAMP despite thepresence of very large amounts of C-terminal βAPP derivatives in thesecells.

These results were confirmed in HEK293 cells over-expressing eitherβAPP_(Swedish) or the SpC99-βAPP cDNA. The latter encodes the C-terminal99 residues of βAPP (corresponding to the products of β-secretasecleavage) plus the βAPP signal peptide. The interaction of PAMP appearsmuch stronger with C99-βAPP than that with C83-βAPP. However, C83-βAPPis much less abundant in these cells (FIG. 6 b, middle panel, lanes1-4). In fact, PAMP does interact with both C99- and C83-βAPP stubs (seeFIG. 6 c, lane 9 and FIG. 8 d). Cumulatively, these results indicatethat PAMP likely interacts with the C-terminal derivatives of βAPP whichare the immediate precursors of Aβ and p3. However, of greater interest,the genotype of the co-expressed PS1 molecule dynamically influenced theinteraction between PAMP and C99-/C83-βAPP stubs. Thus, more C-terminalβAPP fragments co-immunoprecipitated with PAMP in cells expressing theFAD-associated PS1-L392V mutation compared to cells expressing wild typePS1 (and equivalent quantities of nicastrin and C99-βAPP). Conversely,much less C-terminal βAPP derivatives co-immunoprecipitated with PAMP incell lines expressing the loss-of-function PS1-D385A mutation (despitethe presence of very large amounts of C-terminal βAPP derivatives inthese cells). These effects are more easily seen in cellsover-expressing the C99-βAPP construct. However, similar but lesspronounced differences were also observed in cells over-expressingfull-length βAPP_(Swedish). More importantly, the PS1-sequence-relateddifferences in the interaction of PAMP with C-terminal βAPP derivativeswere most evident within 24 hours of transient transfection of PAMP. By72 hours, the PS1-sequence-related differences were largely abolished.This dynamic change in the interaction of PAMP with C99/C83-βAPP was notdue to changes in the levels of PS1, C-terminal βAPP derivatives orPAMP. One interpretation of these results is that the presenilins may bedynamically involved in regulating or loading PAMP with the substratesof γ-secretase.

Presenilin binding domains of PAMP. In transiently transfected cells (inwhich the 7-10 kDa C-terminal of PAMP can be detected), anti-PS1immunoprecipitation products contain both full length PAMP and the ˜7-10kDa C-terminal PAMP fragments. Similarly, in these cells,immunoprecipitation with antibodies to the C-terminus of βAPP (Ab369)also renders C-terminal nicastrin epitopes. The TM domain of PAMP is nothighly conserved in evolution. These results suggest that theC99-/C83-βAPP and PS1/PS2-binding domain(s) of PAMP are in the TM domainor C-terminus.

Discussion

The above results indicate that PAMP is a component of the PS1 and PS2intracellular complexes. The observations that PAMP also binds to theC-terminal fragment of βAPP (arising from α- and β-secretase cleavage offull length βAPP), that the degree of binding of these fragments to PAMPis modulated by mutations in PS1, and that the direction of thismodulation is congruent with the effects of each mutant of Aβ production(i.e., the pathogenic L392V mutation increases binding to PAMP andincreases Aβ₄₂ production whereas the D385A mutation has the oppositeeffects) strongly argues that PAMP is part of a functional complexinvolved in processing of C-terminal βAPP derivatives. Similarly, theobservation that inhibition of PAMP expression in C. elegans leads to aphenotype similar to that of glp/Notch loss of function, argues thatPAMP, like PS1 and PS2, is also a functional component of the pathwaysinvolved in processing of Notch. This conclusion is strengthened by thefact that the murine PAMP gene maps within a 700 kb interval on murinechromosome 1 which carries the Looptail mutant, and is thus likely to bethe site of the Looptail mutation. Looptail has a number of phenotypicsimilarities to those of Notch and PS1 knockouts in mice. BecauseLooptail is a model of human spinal cord malformations including spinabifida, PAMP biology may also provide some useful insights into thisneurological developmental defect as well.

At the current time the exact role of PAMP in thepresenilin-complex-mediated processing of βAPP and Notch-like moleculesis not fully defined. Inspection of the primary amino acid sequence ofPAMP does not reveal very strong homologies to known proteases. However,the recombinant expression systems of the invention permit evaluation ofthree-dimensional structure of PAMP; it is possible that PAMP itself hasa protease activity. However, it is currently more plausible that PAMPplays another role in βAPP and Notch processing. Thus, PAMP may beinvolved in the function of PS1 and PS2 complexes by binding substratesfor γ-secretase. The efficacy of this binding is clearly modulated byPS1 mutations in a direction which is commensurate with the effect ofthese mutations on γ-secretase activity. Alternatively, PAMP may have aregulatory role on the activity of the presenilin complexes. This isconsistent with the observation that residues 625-641 of human andmurine PAMP contain a motif similar to cyclic nucleotide binding domainsof several other unrelated proteins.

Regardless of its precise role, it is clear that PAMP and PS1 both playimportant roles in γ-secretase mediated processing of βAPP. Hence,knowledge of PAMP and its biology will now serve as a target for effortsto manipulate the function of the presenilin complexes in patients withschizophrenia and/or Alzheimer Disease and related disorders, patientswith malignancies (in which the presenilins have been implicated byvirtue of a role in programmed cell death), and in disorders ofdevelopment especially of the spinal cord and brain (in view of theknown effects of PS1 knockout and the strong likelihood that PAMP is thesite of Looptail mutations in mice). In particular, knowledge of thedomains of PAMP involved in binding presenilins and βAPP derivatives(which currently appears to be located within the C-terminaltransmembrane and hydrophilic domains of PAMP) and the identification ofputative ligands interacting with the conserved domains at thehydrophilic N-terminus of PAMP will considerably expedite this goal.

We have found that the strength of the interaction between PAMP and theC-terminal fragments of βAPP (which is the precursor Aβ) is determinedby the genotype at PS1. Thus, clinical mutations in PS1 cause AlzheimerDisease and an increase in the production of Aβ₄₂ are associated withincreased binding of the C-terminal fragments of βAPP to PAMP.Conversely, loss of function mutations in PS1 (Asp385Ala) which inhibitγ-secretase cleavage of C-terminal fragments of βAPP, are associatedwith abolition of the interaction between PAMP and the C-terminalfragments of βAPP.

Finally, the apparent C-terminal proteolytic derivatives of PAMP couldeither be authentic, or simply artefacts due to the V-5 tag. If they areauthentic, this observation raises the possibility that PAMP may undergopost-translational processing events which are potentially similar tothose of βAPP and/or Notch. Three observations support our discovery ofPAMP. First, in contrast to βAPP and Notch, which are not majorconstituents of the high molecular weight presenilin complexes, andwhich can only be inconsistently shown to be directly associated withPS1/PS2, PAMP is a major stoichiometric component of the presenilincomplexes. Second, PAMP selectively interacts only with C-terminalderivatives of βAPP which are substrates for γ-secretase cleavage, andthis interaction is modulated by PS1 mutations in a way which reflectsthe functional consequences of these PS1 mutations. Third, inhibition ofPAMP expression in C. elegans leads to a disease phenotype likely to bein the glp/Notch signaling pathway.

Example 2 PAMP Mutants

Site-directed mutagenesis was used to generate the following artificialmutations in PAMP:

Cys: PAMP_(C230A) in the 4 conserved cystine motif

DYIGS: PAMP_(D336A/Y337A) in the central conserved region

D369L: PAMP_(Δ312-369) in the central conserved region

D340X: PAMP_(Δ312-340) in the central conserved region

YDT: PAMP_(D458A) in the putative ‘aspartyl protease’ DTA site

SPAF: PAMP_(P633A/F635A) in the SPAF motif

TM: PAMP_(S683A) in the TM domain

C3D: PAMP_(Δ630-668) in the conserved region adjacent to the TM domain

To further examine the role of PAMP in βAPP processing, we inserted PAMPcDNAs, harboring the above mutations as well as normal/wild type PAMP(PAMP_(wt)) cDNA and the cDNA for an unrelated protein (LacZ), in frameinto pcDNA6 vectors. A series of HEK293 cell lines stably expressingendogenous PS1, βAPP_(Swedish) and either wild type PAMP or PAMPconstructs in which various conserved domains had been mutated ordeleted, were then created by transfection. PAMP expressing cells wereselected with lasticidin to generate stable cell lines. Conditionedmedia from these cell lines were collected after 6-24 hours and Aβ₄₀ andAβ₄₂ were measured by ELISA.

In the PAMP_(D336A-Y337A) mutant, both Aβ₄₀ and Aβ₄₂ levels wereincreased, and there was also a 68% increase in Aβ₄₂/Aβ₄₀ ratio which isvery similar to that observed in clinical mutations in APP, PS1, andPS2, associated with early onset Alzheimer Disease. The Aβ₄₂/Aβ₄₀ ratiowas also increased in one cell line expressing the PAMP_(C230A) mutant.

In contrast, both the total Aβ₄₂ and Aβ₄₀ levels and the Aβ₄₂/Aβ₄₀ ratiowere massively reduced (to only 18% of the control) in thePAMP_(Δ312-369) mutant. A similar but less profound reduction of boththe total Aβ₄₂ and Aβ₄₀ levels and the Aβ₄₂/Aβ₄₀ ratio was observed inthe conditioned medium from the PAMP_(Δ312-340) cell lines.

There is no apparent difference in Aβ₄₂ or Aβ₄₀ levels, or in theAβ₄₂/Aβ₄₀ ratio, when the PAMP_(wt), PAMP_(D458A), PAMP_(Δ630-668),PAMP_(P633A/F635A), and PAMP_(S683A) cells were compared to controllines (expressing LacZ, or empty vector).

Thus, certain PAMP mutants cause biochemical changes similar to thoseinduced by mutations in the βAPP, PS1, and PS2 genes which give rise toAD, and which may be implicated also in schizophrenia. These artificialPAMP mutations can therefore be used to generate cellular and othermodel systems to design treatments and preventions for schizophrenia, ADand other neurodegenerative and/or neuropsychiatric disorders. Thesemutations also show that PAMP is involved in the pathogenesis of AD, andmay provide information for the design of specific molecular diagnosticsor therapeutics for schizophrenia, AD, and other neurological disorders.

When compared to mock-transfected or LacZ transfected cells,overexpression of wild type PAMP, and overexpression of most PAMPmutation- or deletion-constructs had no significant effect on Aβsecretion. However, missense mutation of the conserved DYIGS motif toAAIGS (residues 336-340 of human PAMP) caused a significant increase inAβ₄₂ secretion, a smaller increase in Aβ₄₀ secretion, and an increase inthe Aβ₄₂/Aβ₄₀ ratio (p<0.001; Table 2). This increase in Aβ₄₂ productionwas equivalent to that of FAD-related missense mutations in PS1.Conversely, deletion of the DYIGS domain in two independent constructs(PAMP_(Δ312-369) and PAMP_(Δ312-340)) caused a significant reduction inboth Aβ₄₂ and Aβ₄₀ secretion which was more profound in PAMP_(Δ312-3369)cells than in PAMP_(Δ312-340) cells (Table 2). The magnitude of thereduction in Aβ secretion in PAMP_(Δ312-369) cells was equivalent tothat observed with the PS1-D385A loss-of-function mutation. Somewhatunexpectedly, and in contrast to PS1^(−/−) and PS1-D385A cells, thereduction in Aβ secretion in NCT_(Δ312-369) and NCT_(Δ312-340) cells wasnot accompanied by the expected accumulation of C99- and C83-βAPP stubs.Since there was no consistent change in the levels of soluble βAPP(βAPP_(s)) in the conditioned medium of any of the PAMP mutant cells,the most probable explanation for this result is that C99- and C83-βAPPstubs which do not enter the PAMP:presenilin complex for γ-secretasecleavage to Aβ may be degraded by other pathways.

The effects of PAMP mutations on Aβ secretion were not due to trivialexplanations such as differences in the levels of PAMP, βAPPholoprotein, or PS1/PS2. None of these mutations caused any consistent,detectable change in the amount of APP_(s) in conditioned medium or inthe amount of C99/C83βAPP that could be co-immunoprecipitated with PAMP.However, both the PAMP_(Δ312-369) mutant and the PAMP_(Δ312-340)deletion mutant significantly reduced the amount of PS1 which could beco-immunoprecipitated with PAMP. Interestingly, the reduction inefficiency of binding to PS1 was proportional to the reduction in Aβsecretion induced by each deletion mutant. Multiple mechanismsunderlying the effect of mutations in the first conserved domain canexplain these results. This domain contains no obvious functional motifs(e.g., for glycosylation etc.), nor does it have significant sequencehomology to other known proteins. Consequently, the three functionallyactive PAMP mutations either affect a presenilin-binding domain in PAMP,or affect a specific regulatory domain of PAMP which modulates bothdirect binding of PAMP to PS1 and the subsequent γ-secretase-mediatedcleavage of PAMP-bound C99- and C83-βAPP stubs. TABLE 2 Aβ₄₂/Aβ₄₀Transfection Normalized Aβ₄₂ Normalized Aβ₄₀ Ratio Mock (LacZ/empty 1.01.0 1.0 vector) wild type PAMP 1.03 ± 0.09 1.05 ± 0.07 0.99 ± 0.07D336A/Y337A 3.09 ± 0.59 1.61 ± 0.19 1.81 ± 0.15 (p < 0.001) (p = 0.001)(p < 0.001) PAMP_(Δ312-369) 0.05 ± 0.04 0.31 ± 0.06 0.09 ± 0.05 (p <0.001) (p < 0.001) (p < 0.001) PAMP_(Δ312-340) 0.33 ± 0.04 0.55 ± 0.040.59 ± 0.06 (p = 0.002) (p = 0.001) (p = 0.003)

Example 3 PAMP Interaction with Notch

PAMP interaction with Notch was studied using a Notch-cleavage assay (DeStrooper, 1999). Notch cDNA was tagged with myc to the membrane-portionof Notch or to the soluble proteolytic derivative called Notchintra-cellular domain (NICD). V5-epitope-tagged PAMP andmyc-tagged-Notch cDNAs were co-transfected into HEK293 cells.Thereafter, V5-tagged-PAMP was immunoprecipitated withanti-V5-antibodies, and the immunoprecipitation products investigatedfor myc-tagged proteins. In the immunoprecipitate, myc-tagged-Notch wasfound, but not myc-tagged-NICD. This result indicates a specificinteraction between PAMP and the Notch precursor (which is the expectedsubstrate for presenilin-dependent S3 cleavage). In contrast, PAMP didnot bind to NICD, which arises as a product of presenilin-PAMP-mediatedS3 cleavage of the Notch precursor.

Example 4 PAMP Screening of Schizophrenia Patients

A study is conducted to investigate PAMP sequence, its expressionlevels, and its activity, in selected study objects. Initially, thestudy objects are selected from families having (1) increased rates ofschizophrenia, and (2) a high proportion linked to the susceptibilitylocus on chromosome 1 q21-q22 as described in Brzustowicz et al., 2000.Control individuals are selected from families with no or only a rareoccurrence of schizophrenia.

Tissue samples are collected from study objects and control objects. Thesamples can be obtained either by sampling tissue fluids such as bloodand cerebrospinal fluid, or by taking biopsies from selected tissues. Incertain instances it may be preferable to collect tissue biopsies, e.g.from brain, kidney, or lung, from deceased study or control objects,i.e. post-mortem.

The samples are analyzed for at least one of the following: (1) sequenceof the entire or selected portions of the PAMP gene; (2) sequence andlevels of PAMP mRNA; (3) sequence, levels, and activity of PAMP protein;(4) levels of a PAMP substrate. Identification of relevant mutations inthe PAMP gene or mRNA is performed by using PCR together with primersspecific for PAMP DNA or mRNA and radiolabeled nucleotides,hybridization analysis, and/or other automated sequencing techniquesdescribed herein or in references provided in the present disclosure,which are all incorporated by reference. Mutations in and levels of thePAMP protein is studied by, e.g., purifying PAMP from the tissue sample,performing enzyme-linked immunosorbent assay (ELISA) or otherquantitative or semi-quantitative immunoassays, Edman degradationanalysis, mass-spectroscopy, Western blotting, or other analyticaltechniques described herein or in references in the present disclosure.PAMP biological activity assays are conducted as described herein byeither in vivo methods (e.g., monitoring βAPP processing and theproduction of amyloid-β peptide (Aβ), or other suitable proteinsubstrates for PAMP including Notch, etc.), or by in vitro assays (usingeither whole cell or cell-free assays to measure processing of suitablesubstrates including βAPP or parts thereof, and other proteins such asNotch).

The results from these assays will preferably show any significantcorrelation between mutations in and/or expression levels of PAMP or thePAMP gene and susceptibility to schizophrenia. PAMP or PAMP mutations,or altered PAMP or PAMP levels, identified in this manner canadvantageously be used in the creation of in vivo assays (e.g.,transgenic animals) or in vitro assays to study induction and/orprogression of schizophrenia, as well as in the screening of potentialtherapeutic agents for schizophrenia. For instance, in an in vivotransgenic/recombinant mouse model, partial phenotypes could be examinedvia behavioral deficits in, e.g., exploratory behavior, novelty seeking,cognitive flexibility/rigidity, sensitivity to dopamine-induced motordisturbances, etc. (see Cloninger et al., 1996).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that values are approximate, and areprovided for description.

Patents, patent applications, and publications are cited throughout thisapplication, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

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PATENT LITERATURE

-   Canadian Patent Application No. 2,012,311-   European Patent Publication No. EP 453242-   European Patent Publication No. EP 488528-   European Patent Publication No. EP178220-   European Patent Publication No. EP 185 573-   International Patent Publication No. WO 96/34099-   International Patent Publication No. WO 95/02697-   International Patent Application No. WO 95/22617-   International Patent Publication No. WO 96/22378-   International Patent Application No. WO 91/09967-   International Patent Publication No. WO 89/12690-   International Patent Publication No. WO95/02697-   International Patent Publication No. WO94/12649-   International Patent Publication No. WO 91/18088-   International Patent Publication No. WO94/28152-   International Patent Application No. WO94/26914-   International Patent Publication No. WO 92/00252-   International Patent Publication No. WO94/28938-   International Patent Publication No. WO 9428028-   International Patent Publication No. WO 99/01175-   International Patent Publication No. WO 99/01158-   International Patent Publication No. WO 99/01157-   International Patent Publication No. WO 95/07358-   International Patent Application No. WO 95/28494-   International Patent Publication No. WO 93/09239-   International Patent Publication No. WO95/21931-   International Patent Publication No. WO96/25508-   International Patent Publication No. WO95/21931-   International Patent Publication No. WO96/17823-   International Patent Publication No. WO95/18863-   International Patent Publication No. WO94/26914-   International Patent Application No. WO 90/02806-   International Patent Publication No. WO 94/21807-   International Patent Application No. WO 89/07150-   International Patent Application No. WO 96/39036-   International Patent Application No. WO 97/19182-   International Patent Publication No. WO 92/05263-   International Patent Application No. WO 95/26411-   U.S. Pat. No. 5,986,054-   U.S. Pat. No. 5,040,540-   U.S. Pat. No. 6,020,143-   U.S. Pat. No. 5,654,168-   U.S. Pat. No. 5,124,263-   U.S. Pat. No. 5,010,175-   U.S. Pat. No. 4,631,211-   U.S. Pat. No. 4,980,289-   U.S. Pat. No. 4,959,317-   U.S. Pat. No. 4,946,778-   U.S. Pat. No. 5,132,405-   U.S. Pat. No. 5,777,195-   U.S. Pat. No. 5,476,786-   U.S. Pat. No. 4,650,764-   U.S. Pat. No. 5,399,346-   U.S. Pat. No. 5,225,539-   U.S. Pat. No. 5,801,030-   U.S. Pat. No. 5,693,762-   U.S. Pat. No. 5,693,761-   U.S. Pat. No. 5,139,941-   U.S. Pat. No. 5,585,089-   U.S. Pat. No. 4,797,368-   U.S. Pat. No. 5,616,491-   U.S. Pat. No. 4,861,719-   U.S. Pat. No. 5,459,127.-   U.S. Pat. No. 5,792,844-   U.S. Pat. No. 5,814,500-   U.S. Pat. No. 5,811,234-   U.S. Pat. No. 5,780,607-   U.S. Pat. No. 5,677,437-   U.S. Pat. No. 5,034,506-   U.S. Pat. No. 5,783,682-   U.S. Pat. No. 5,580,859-   U.S. Pat. No. 5,589,466-   U.S. Pat. No. 5,637,684

1. A method for detecting a mutation in presenilin associated membraneprotein (PAMP) associated with a neuropsychiatric or neurodevelopmentaldisorder, which method comprises detecting a variation in a sequence ofa gene encoding PAMP obtained from an individual diagnosed with orsuspected of having said disorder.
 2. The method of claim 1, wherein thedisorder is schizophrenia.
 3. A method for diagnosing individualspredisposed to or having a neuropsychiatric or neurodevelopmentaldisorder, which method comprises detecting a mutation in a gene encodingPAMP obtained from an individual.
 4. The method of claim 3, wherein thedisorder is schizophrenia.
 5. The method according to claim 3, whereindetection of the mutation comprises measuring a level of transcriptionalactivity of the gene.
 6. he method according to claim 3, whereindetection of the mutation comprises measuring PAMP activity.
 7. Themethod of claim 6, wherein said PAMP activity comprises PAMP expressionlevel or activity of a product of a PAMP modified substrate.
 8. A methodfor identifying a compound that is useful in treating a neuropsychiatricor neurodevelopmental disorder, which method comprises detectingmodulation of of PAMP expression in a transgenic animal that expressesPAMP, wherein the animal is contacted with the compound.
 9. The methodof claim 8, wherein the disorder is schizophrenia.
 10. (canceled) 11.(canceled)
 12. A method for identifying a compound that is useful intreating a neuropsychiatric or neurodevelopmental disorder, which methodcomprises: (a) contacting a complex between a presenilin associatedmembrane protein (PAMP) and an agent, which agent provides a detectableconformational or functional change in said PAMP upon interaction with asubstance being analyzed for activity against a neurodegenerativedisease, with a test compound; and (b) detecting a conformational orfunctional change in PAMP in the complex.
 13. The method of claim 12,wherein the disorder is schizophrenia.
 14. The method of claim 12,wherein the test compound is a protein that interacts with PAMP.
 15. Amethod for treating neuropsychiatric or neurodevelopmental disorder in amammalian host which expresses at least one PAMP protein or a naturallyoccurring variant, which method comprises administering to the host anamount of compound effective to modulate PAMP expression in the host.16. The method of claim 15, wherein the disorder is schizophrenia.